Abstract

Purpose: Microphthalmia and anophthalmia
are at the severe end of the spectrum of abnormalities in ocular
development. A few genes (orthodenticle homeobox 2 [OTX2],
retina and anterior neural fold homeobox [RAX], SRY-box 2 [SOX2],
CEH10
homeodomain-containing homolog [CHX10], and growth
differentiation factor 6 [GDF6]) have been implicated mainly in
isolated micro/anophthalmia but causative mutations of these genes
explain less than a quarter of these developmental defects. The
essential role of the LIM homeobox 2 (LHX2) transcription factor in
early eye development has recently been documented. We postulated that
mutations in this gene could lead to micro/anophthalmia, and thus
performed molecular screening of its sequence in patients having
micro/anophthalmia.

Methods: Seventy patients having
non-syndromic forms of colobomatous microphthalmia (n=25), isolated
microphthalmia (n=18), or anophthalmia (n=17), and syndromic forms of
micro/anophthalmia (n=10) were included in this study after negative
molecular screening for OTX2, RAX, SOX2, and CHX10
mutations. Mutation screening of LHX2 was performed by direct
sequencing of the coding sequences and intron/exon boundaries.

Results: Two heterozygous variants of
unknown significance (c.128C>G [p.Pro43Arg]; c.776C>A
[p.Pro259Gln]) were identified in LHX2 among the 70 patients.
These variations were not identified in a panel of 100 control patients
of mixed origins. The variation c.776C>A (p.Pro259Gln) was
considered as non pathogenic by in silico analysis, while the variation
c.128C>G (p.Pro43Arg) considered as deleterious by in silico
analysis and was inherited from the asymptomatic father.

Conclusions: Mutations in LHX2
do not represent a frequent cause of micro/anophthalmia.

Microphthalmia and anophthalmia are at the severe end of the
spectrum of abnormalities in ocular development. The combined
occurrence rate for these two malformations is estimated between 3 and
30 per 100,000 births [1].
Mutations
in several genes have been found in syndromic and
non-syndromic anophthalmia. Heterozygous mutations in SRY-box 2 (SOX2)
account
for approximately 10% of anophthalmias [2,3].
Growth
differentiation factor 6 (GDF6) mutations may account for up to
8% of micro/anophthalmia [4,5]. Other genes have
been identified as causing isolated anophthalmia or microphthalmia in
humans (orthodenticle homeobox 2 [OTX2], retina and anterior
neural fold homeobox [RAX], and CEH10 homeodomain-containing
homolog [CHX10]) [1,3,6-8]. These latter are
implicated in a very small proportion of affected individuals, implying
wide genetic heterogeneity to match the phenotypic variability.

The LIM homeobox 2 (LHX2) transcription factor has been shown to be
essential for mammalian eye development and mice deficient in
functional Lhx2 protein have been shown to display anophthalmia [9]. More recent studies
on mouse models have demonstrated that, during eye development, Lhx2
regulates levels and/or expression patterns of Rax, Chx10, Sox2, and
Otx2 [10,11], themselves
involved in human micro/anophthalmia.

We hypothesize that mutations in LHX2 could lead to severe
eye developmental disorders including micro/anophthalmia. We thus
performed molecular analysis in 70 micro/anophthalmia patients for whom
previous molecular analysis of genes implicated in isolated
micro/anophthalmia (SOX2, OTX2, RAX, and CHX10)
failed
to identify any causative mutation.

Patients

Seventy patients having non-syndromic forms of colobomatous
microphthalmia (n=25), isolated microphthalmia (n=18), or anophthalmia
(n=17), or syndromic forms of micro/anophthalmia (n=10) were included
in this study. Their informed consent was obtained beforehand,
according to French law. Micro/anophthalmia was considered as syndromic
when the patient presents at least one other non ocular malformation
(in our patients, associated malformations were intestinal atresia
multiple, corpus callosum agenesis, heart malformation, deafness, Dandy
Walker malformation, labiopalatal cleft, sexual ambiguity, hypospadias,
arthrogryposis, and choanal atresia). All patients included had
undergone molecular analysis of SOX2, OTX2, RAX,
CHX10. Direct sequencing of the coding regions and exon/intron
boundaries and exclusion of exonic rearrangement by Quantitative
Multiplex PCR of Short fluorescent Fragments (QMPSF) of these genes
failed to identify any causative mutation in these patients.

Techniques

The 5 exons of the LHX2 gene were amplified by PCR using
primers deduced from the LHX2 genomic sequence. Primer pairs
and PCR conditions used are summarized in Table 1. Products were
amplified in 25 µl reactions containing 50 ng genomic DNA, 1× PCR
buffer, 0.2 mM dNTPs, 2 mM MgCl2, 100 nM forward
primer, 100 nM reverse primer, and 1 U of Taq polymerase. Betaine (1 M)
was added in PCR mix for exons 1 and 2. All PCR reactions were
performed with a 5 min 95 °C denaturing step, followed by 14
cycles of 95 °C for 30 s, annealing temperature for 30 s
(70 °C to 62 °C, −0.5 °C/cycle) and 72 °C for 45 s,
followed by 20 cycles of 95 °C for 30 s, 62 °C for 30 s, and
72 °C for 45 s with a final elongation step of 72 °C for 7
min. PCR amplifications were subsequently purified using QIAquick Gel
Extraction kit (QIAGEN SA, Courtaboeuf, France), and both forward and
reverse strands were sequenced using Big Dye DNA sequencing kit
(Applied Biosystems, Warrington, UK). Reactions were analyzed in an
ABI3100 sequencer (Applied Biosystems). Sequence variations were
numbered with the adenine of the ATG initiation codon as the first
nucleotide (the LHX2 GenBank accession number was NM_004789.3).

LHX2 encodes the transcription factor LHX2 which is highly
conserved across species [12]
and
has recently been demonstrated to play a critical role in eye
development [10,11]. LHX2 is required
to induce or maintain expression of genes required at the early optic
vesicle stage for regionalization, establishment of retinal
dorsoventral polarity, retinal progenitor cell properties, and lens
specification [11].
LHX2
has thus been proposed to link the multiple pathways needed for
transition of the optic vesicle to the optic cup [11]. Mice lacking Lhx2
expression display anophthalmia, and this transcription factor has been
involved in regulation of expression levels and/or expression patterns
of genes already involved in micro/anophthalmia (SOX2, RAX,
CHX10, and OTX2) during eye development [10,11]. We hypothesized
that mutations in LHX2 may be involved in human
micro/anophthalmia, and thus molecular screening of this gene in 70
micro/anophthalmic patients was performed.

Molecular analysis allowed identification of three sequence
variations. We observed presence of the described SNP c.783G>C
(p.Pro261Pro) either in a heterozygous or homozygous state in 42 out of
70 patients. In addition, two variants of unknown significance were
identified. The heterozygous c.128C>G (p.Pro43Arg; Figure 1A)
was
identified in an anophthalmic patient originating from Libya for
whom no other sequence variation was found. Pro43 amino-acid is
conserved among species (Figure 1C) and is located
closely to the conserved LIM domain 1 of the protein. This variation
was not identified in a panel of 200 control chromosomes of mixed
geographical origins (Caucasian, African, and Asian), and was
considered as probably damaging by in silico analysis (PolyPhen and SIFT software). However, familial
study has shown that this variation was inherited from his father who
harbors a normal ocular examination. Thus, this heterozygous variation
may be non-pathogenic, even if dominant inheritance with incomplete
penetrance can not be totally ruled out. We cannot also exclude
presence of an undetected maternally inherited mutation (e.g located in
intronic or promoter region sequences, or an exonic rearrangement)
fitting with an autosomal recessive inheritance. In a French patient
with colobomatous microphthalmia, we identified the heterozygous
variation c.776C>A (p.Pro259Gln; Figure 1B). This variation was
not identified in a panel of 200 chromosomes from Caucasian controls.
No sample was available for his parents, and familial segregation study
was not possible. Additionally, there are arguments against the
implication of this variation in the patient’s ocular phenotype. First,
Pro259 is not a conserved amino-acid among species (Figure 1D),
and
a glutamine is present at this position in several distant species
including Xenopus, Chicken, and Fugu Fish [12]. Second, this
variation was considered as non damaging by in silico analysis (PolyPhen and SIFT software). Thus, we consider
this variant as probably non deleterious. Molecular analysis failed to
identify any other sequence variation in the remaining 68 patients
included in this study.

In conclusion, although mutations in LHX2 may nevertheless
be implicated in some micro/anophthalmia patients, our results suggest
that such sequence variations are not a frequent cause of
micro/anophthalmia. Molecular basis of these ocular malformations
remains still poorly understood and further work remains to be achieved
to identify new micro/anophthalmia genes.

This work was supported by a grant from the Clinical Research
Hospital Program from the French Ministry of Health (PHRC 09 109
01) and by Retina France. The authors are grateful to Jacqueline
Butterworth for help in preparing the manuscript.